ABSTRACT The precise molecular targets of ethanol action in the brain have remained elusive for decades, primarily as a result of the transient and low-affinity nature of ethanol interactions with specific proteins or other cellular components. The endogenous opioid system has been implicated in the rewarding, reinforcing, motivational, and neurochemical effects of drugs of abuse, including ethanol. We have previously demonstrated that acute ethanol administration in rats significantly increases extracellular endorphin levels in the nucleus accumbens (NAc). We have also generated novel preliminary data indicating that immunoneutralization of ?-endorphin in the NAc shell subregion, but not the core, attenuates low dose ethanol self-administration. These findings suggest that endorphinergic transmission in the NAc shell contributes to the motivational effects of low dose ethanol. Finally, we utilized transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the promoter for the ?-endorphin precursor peptide pro-opiomelanocortin (POMC), to assess the potential activation of these neurons by low dose ethanol. We observed that in these POMC-EGFP mice, low dose ethanol consumption in the drinking-in-the-dark paradigm resulted in brain and blood ethanol levels <10 mM, and activated POMC-expressing neurons in the arcuate nucleus of the hypothalamus (ArcN). Collectively, these data indicate that brain POMC/endorphin systems are molecular targets of low dose ethanol. However, additional studies are needed to test our overarching hypothesis that ArcN POMC-expressing neurons are a target of low dose ethanol in the brain. To accomplish this, we have formulated the following inter-related yet independent Specific Aims. In Aim 1, we will further characterize the ability of low dose ethanol to activate ArcN POMC-expressing neurons. POMC-EGFP mice will be tested in the drinking-in-dark paradigm to determine the dose-dependency, specificity, and desensitization of ethanol-induced activation of ArcN POMC-expressing neurons. In Aim 2, we will determine the accumbal targets of ArcN POMC-expressing neurons activated by low dose ethanol. This will be accomplished by the use of Cre-dependent viral retrograde tracing methods in POMC- Cre mice. Finally, in Aim 3, we will determine the molecular adaptations in ArcN POMC-expressing neurons that occur following low dose ethanol consumption. This will be accomplished by utilizing POMC-Cre Ribotag mice to determine cell-type specific changes in RNA expression of biologically related genes induced by low dose ethanol consumption. To increase the translational impact of these studies, all proposed experiments will utilize both male and female subjects, and ethanol intake procedures that allow voluntary consumption as opposed to passive administration by an experimenter. Together, these studies will ultimately guide the development of pharmacological or other approaches for inactivating the effects of ethanol in the brain, which may ultimately reduce the medical, socioeconomic and legal costs of ethanol abuse to society.